This semester Prof. Isaacson and I are working with HEK 293 cells instead of 293t cells. In the past, we worked with 293 t cell line and study the localization of the TORC family and tax protein in these. HEK 293 is a cell line derived from human embryonic kidney cells in culture and they differ from the 293t cells because they do not contain the SV40 large T antigen that is capable of inducing malignant transformation. The purpose of working with the HEK 293 this semester is to study localization of the TORC family in the cells and afterwards compare it to the localization of these proteins in the 293 t cells. We are interested in seeing if malignant transformation of the cells affects the localization of the proteins (TORC).
We are currently doing immunofluorescence to study localization but so far this semester they have all been a fail (3 so far). The HEK 293 cells do not grow as fast as the 293 t cells and do not adhere as well either. This has proven to be a problem as they are harder to maintain after every split and they do not adhere to the glass cover slips very well. In fact, I had to dispose of two plates because they did not adhere at all to the glass cover slips. If they do not adhere, I am not able to perform any experiment on them and this has been my biggest challenge this semester. I am hoping that by treating the glass cover slips with a special chemical the cells will be able to adhere and I will be able to perform my experiments.
As part of my research I will be working with 293T cells and analyzing TORC and Tax localization in the cells. I will also be analyzing the localization of Tax and TORC in 293 cells and than comparing both of the localization in normal vs. cancerous cells. The main goal of the research is to analyze the gene expression of HTLV in the presence of tax and cellular transcription factors, which are thought to enhance HTLV gene expression. HTVL is the first retrovirus that was discovered and it is known to develop an often rapidly fatal leukemia, debilitative myelopathy, uveitis, dermatitis, and inflammatory disorder among others. The goal of this academic year is to finalize our findings and determine if TORC 3 enhances HTLV gene expression.
To study localization we will be using immunofluorescence technique. For this technique we use antibodies that are specific to tax and TORC 3. Each antibody will emit a specific wavelength of light once excited allowing us to view it under the microscope. The light is specific to the antibody (red or green) and is only emitted when the antibody is present in the cells, indicating the presence of the protein of interest in the cells. Prior to staining, we will transfect cells with Tax and TORC, stain with the antibody, and view the slides under a special microscope. To transfect cells we must grow them in a special media and incubate them in a regulated environment to ensure their growth occurs at optimal conditions. Many times many trials of immunofluorescence are needed to obtain pictures that are of good quality. I look forward to all the exciting new challenges this academic year will bring.